Gs junior titanium empcr kit lib l

The V4 hypervariable region of the 16S ribosomal RNA gene was amplified using GS FLX 454 one way read barcoded fusion primers (Lib-L kit, Primer A, Primer B, Roche 454 Life Science, Branford, CT, USA) with the template specific sequences F515 and R806 (Caporaso et al., 2011a) . PCR triplicates were run using illustra Hot Start Mix RTG beads (GE Healthcare, Little Chalfont, Bucks, UK) following the manufacturer's protocol with 0.4µM of each primer and 1µL of DNA extract. The PCR triplicates were pooled and purified with the QiaQuick Gel Extraction kit (Qiagen, Venlo, Netherlands). After DNA quantification with QuantiFluor dsDNA Dye (Promega, Madison, WI, USA) the samples were pooled equimolar and were run on a 2100 Bioanalyzer (Agilent, Waldbronn, Germany) for quality assessment and quantification. Emulsion PCR of pooled samples was performed with the GS Junior Titanium emPCR Kit (Lib-L) (Roche 454 Life Science, Branford, CT, USA) as per the manufacturer's instructions. Sequencing was run on a GS Junior instrument using GS Junior Titanium Sequencing Kit and PicoTiterPlate Kit (Roche Diagnostics, Basel, Switzerland).

SCN1A amplicons of each patient were pooled at equal molecule amounts and purified by MinElute DNA purification kit (Qiagen, ABD). Ten nucleotides long MID sequences specifying each patient were ligated to amplicons in each pool using GS Rapid Library Preperation Kit Lib-L (Roche, Germany) as described in the GS Junior Rapid Library Preparation Manual. Concentration of pooled amplicons was measured both by Quant-iT-PicoGreen dsDNA assay Kit (Invitrogen, ABD) and also by qPCR using KAPA NGS quantification kit (KAPA systems, ABD) on LightCycler 480II. Dilutions were made to have single fragment per bead and the sequencing library was prepared for emulsion PCR (emPCR) using GS Junior Titanium emPCR Kit Lib-L (Roche, Germany) as described in GS Junior emPCR Lib-L manual. DNA attached beads were picked up magnetically and pyrosequenced using GS Junior sequencing kit by following the instructions in the GS Junior sequencing method manual. Amplicon sequences were analyzed by Amplicon Variant Analyzer (AVA) program (Roche, Gernmany) using the SCN1A reference sequence, PCR primer and MID sequence information. In the result of AVA analysis, a variant list was obtained for each patient. The variants were filtered for known SNPs and unique variants were validated by Sanger sequencing.